Abstract: OBJECTIVE To establish the present study of a quantitative analysis method to determine the concentration of free fatty acids(FFAs) in serum by gas chromatography-mass spectrometry(GC-MS). To investigate the correlation between the FFAs levels and hyperlipidemia in serum and the intervention effect of simvastatin. METHODS Derivatization was used to obtain methyl product. High-fat diet was used to induce hyperlipidemia animal model. The positive group was treated with simvastatin for four weeks. Meanwhile, the analysis method of serum FFAs was established by GC-MS to obtain the concentration of FFAs. RESULTS The GC-MS method was successfully established and validated for measuring the concentration of FFAs in serum. The results found that the nineteen FFAs were detected in serum of animals, such as caprylic acid, tridecylic acid, linoleic acid, etc. Simvastatin had a significant improvement effect on fourteen serum FFAs. CONCLUSION The content of FFAs in the serum is closely related to hyperlipidemia. Simvastatin can significantly ameliorate the distribution condition serum FFAs, which can provide a scientific basis for the early detection and prognosis evaluation of lipid metabolism-related diseases.